The objective of the proposed research is to recover the optical absorption coefficient for in vivo quantitative photoacoustic imaging (PAI) of biological tissue. PAI can image intact biological tissues with optical absorption contrast at high spatial resolution beyond the optical quasi-ballistic regime (~1 mm). Since optical absorption is sensitive to physiological parameters such as the total concentration and oxygen saturation of hemoglobin, PAI can provide functional imaging. With the aid of functionalized contrast agents (molecular probes), PAI can also provide molecular imaging. PAI has potentially broad applications in both small-animal research and clinical practice, including, for example, (1) the animal study of angiogenesis, brain function, drug discovery, and molecular cell biology, (2) the clinical diagnosis of skin cancer, cervical cancer, and breast cancer, and (3) the intra-operative demarcation of brain cancer and function. Although optical contrast is high, conventional high-resolution optical imaging modalities cannot penetrate more than ~1 mm in scattering biological tissue, a fundamental limit for high-resolution pure optical imaging owing to strong scattering. PAI, combining optical contrast with ultrasonic resolution, breaks through the 1-mm depth limit and provides high-resolution optical imaging, as demonstrated by the applicants'works published in Nature Biotechnology. No other optical technology can image blood vessels and functions at this resolution and depth. PAI is also free of speckle artifacts, which exist in optical coherence tomography and ultrasonography. Furthermore, the maximum imaging depth and spatial resolution of PAI can be scaled with the ultrasonic parameters. Some potential applications were highlighted by Nature Reviews Drug Discovery and Nature Reviews Molecular Cell Biology. PAI directly measures, however, specific optical absorption (absorbed energy per unit volume) rather than absorption coefficient;the former is the product of the latter and the local fluence. Quantitative PAI currently depends on ex vivo or invasive estimation of fluence so that tissue-intrinsic optical absorption coefficient can be retrieved. Central to this proposed research is to develop non-invasive in situ estimation of fluence. Two complementary forms of quantitative PAI are to be investigated: reconstruction-based photoacoustic tomography (PAT) and direct-imaging photoacoustic microscopy (PAM). In this proposed technology-driven research, we will focus on the following specific aims: (1) To quantify optical fluence for quantitative PAT using diffuse optical tomography (DOT);(2) To quantify optical fluence for quantitative PAT using transport optical tomography (TOT);(3) To quantify optical fluence for quantitative PAM using TOT;(4) To validate and evaluate the quantitative PAI systems with tissue phantoms;and (5) To validate and evaluate the quantitative PAI systems in vivo.